The protein P68/BIGH3 (p68) is secreted to the extracellular space of corneal cells. This protein has two clusters of basic amino acids, an Arg- Gly-Asp sequence and fasciclin 1-like repeats. Each of these features are found in proteins that function in cell and axon adhesion and migration. These structural traits in p68 have led to the hypothesis that p68 plays important roles in corneal cell adhesion and migration. Protein fractionation experiments suggest p68 interacts with type VI collagen and microfibril proteins, suggesting it may organize extracellular matrix (ECM) molecules, a fundamental property of healthy, transparent cornea. Interestingly, patients with any one of five p68 autosomal dominant mutations develop corneal dystrophies resulting in reduced transparency and blindness. The mutations result in the amino acid changes R124C, R124H, P501T, R555Q and R555W. These mutations result in the amino acid changes R124C, R124H, P501T, dystrophies Lattice type I, Combined granular-lattice, Lattice type IIIA. Reis-Bucklers and Groenouw type I, respectively. These dystrophies are partly characterized by p68-containing depositions within the cornea. This has led to a second hypothesis that p68 is a causative in the formation of corneal depositions, leading to partial or complete blindness. Currently, limited, if any, information is available about the function of normal p68 in vision, or about the mechanism by which p68 contributes to the formation of corneal depositions. The objective of this project is to test for physiopathologic properties of p68. The first specific aim is to test recombinant p68 substrate to determine whether it may function in wound healing, supporting corneal cell adhesion and migration and neurite extension. Activity will be documented by quantifying cell adhesion. The second aim is to introduce neurite extension. Activity will be documented by quantifying cell adhesion. The second specific aim is to introduce point mutations into basic residue clusters and into the Ar-Gly-Asp site and to delete fasiclin 1-like repeats. These mutants will be tested for altered adhesion properties. A third aim is to introduce each of the corneal dystrophy-related mutations into p68 cDNA, generating each of the five different mutants. These mutants likewise will be tested in cell adhesion experiments. Also, keratocytes will be co-transfected with a plasmid containing epitope-tagged wild type p68 and a plasmid that contains one of the five mutant p68 proteins with a different mutant p68 will be tested for immunodetection and radioimmunoassays. Finally, corneal dystrophy- related mutant p68 proteins will be tested for altered binding with corneal cells and corneal ECM molecules. The results of these experiments should provide new information that will lead to a better understanding of the physiopathologic function of p68 and new disease to help treat patients with these corneal dystrophies.